Our efforts to study HIV-1 protease structure and function in the current funding period have yielded exciting results that form the basis for a new hypothesis, that there is a structural pre-organization of the Gag/Pol protein that brings the p2/nucleocapsid (NC) cleavage site in close proximity to the active site of the protease dimer (PR). The efficiency and order of processing can be influenced by mutations within the region from p2 through p6Pol. In the new Experimental Plan, we will explore the interactions between upstream regions of Gag and PR. In addition, we will extend our analyses of protease structure and function to subtypes other than subtype B. Specific Aim 1 will focus on the influence of mutations within the Gag/Pol fusion protein on the efficiency and order of processing. Our current work has identified several points within the region from the start of protein p2 through the end of the p6Pol [HXB2 amino acids 364-440] where mutations affect processing. We will use a selective mutagenesis method on the full-length gag/pol gene to identify other points of importance within the limits of amino acids 364-440. We will employ an in vitro transcription/translation system to analyze the effects of mutagenesis. Specific Aim 2 will study the Gag-Pol polyprotein processing and examine the properties of the proteases from a variety of subtypes including A2, C, D, H, and F, and recombinant forms A/G, A/C, and B/F, which we have obtained from the NTH AIDS Research & Reference Reagent Program. We will use the same approach as in Specific Aim 1 to examine the rate of and intermediates in polyprotein processing of variants derived from the non-B subtypes. This will provide more natural variants of the Gag-Pol polvprotein sequence and expand our understanding of the consequences of sequence variation. The region of Gag/Pol between matrix (MA) and PR from the non-B subtypes will be placed into our recombinant virus system for analyses of growth to complement the biochemical and structural studies. The presence of a variety of different forms of the virus globally demand that we evaluate the properties of proteases from subtypes other than subtype B, which is predominant in the US and has been used in studies of resistance development,. We will begin with subtypes C, A2, and H. The genes will be subcloned, expressed and purified, and analyzed for kinetic properties, inhibitor binding using clinically-approved drugs, and three-dimensional structure in complex with a variety of the same inhibitors. Specific Aim 3 will explore the properties and the structure of a variety of extended forms of HIV-1 protease, with increasingly longer sequences upstream of the protease, as we know that the initial steps in processing occur in the region upstream of protease. We hypothesize that there is a structural pre-organization of Gag/Pol that brings the C cleavage site [p2/NC] near the active site cleft of the protease, resulting in the initial rapid cleavage of that junction. If this structure can be observed by crystallography, through the construction of an inactive protease-Gag fusion protein, then we will identify a new target for drug discovery, i.e., the interaction surface for formation of the pre-cleavage structure. In addition, we will explore the potential of interactions between the wild-type and mutant forms of protease from Specific Aim 1 and the NC protein as alternative targets for drug discovery.